The present invention relates to an optical system for exciting and measuring fluorescence on or in samples treated with fluorescent pigments. Such optical systems are known, for example, as scanning light microscopes.
Scanning light microscopes have been known for several decades. Their functional principal is based on a light beam being concentrated to a small point of light (the first focal point) on a sample. The sample and this point of light are mutually moved in such a way that a specific area of the sample is scanned (rasterized) by the point of light. The light which penetrates the sample or is reflected by it and/or the fluorescence triggered on or in the sample during the scanning is therefore referred to as “light originating from the sample” and is measured by one or more photodetectors. An enlarged image is produced in that an original measurement point is assigned a specific area on an image of the sample. In principle, such a scanning light microscope therefore includes:                a light source, which produces a light beam;        a sample holder for holding the sample;        an optic for producing a first focal point on the sample;        an optical arrangement for imaging a second focal point using the light which shines through the sample and/or is reflected by the sample and/or which represents fluorescence triggered on or in the sample;        a photodetector for measuring the intensity of the second focal point; and        a scanning mechanism for mutual movement of the sample and first focal point.        
In a conventional scanning light microscope, the light beam is deflected in the direction of the two spatial axes X and Y to illuminate the sample. This procedure hides the disadvantage that the angle of incidence on the sample of the light refracted by the projective lens varies and produces an aberration in the imaging of the sample light by the objective lens. This aberration may be corrected through an appropriate construction of the objective lens. such a lens disadvantageously makes the optic more costly and simultaneously has a limiting effect in regard to the light collecting efficiency and operating distance.
According to U.S. Pat. No. 5,081,350, this problem is solved in that a device is disclosed therein using which the sample is scanned by a light beam. In this case, the device for illuminating the sample and the device for measuring the signal coming from the sample are mounted on a unit which is movable back and forth. The sample is mounted on a sample table movable perpendicularly to this oscillation in this case, so that scanning of the sample is possible with a constant angle of incidence of the illumination. Because, especially for the application of a rapid scanning method, the light source is preferably to be positioned outside the movable part of the scanning light microscope, in this case the use of glass fiber waveguides is suggested, which optically connect the light source to the projective. However, there is the danger that this glass fiber cable may be damaged by the frequent and rapid back and forth movement.
An improved device according to the species is known from U.S. Pat. No. 5,260,569, which solves the problems of the related art described above in that a scanning light microscope is suggested therein which, as a light source, includes a laser, a mirror for deflecting the light, coming out of the laser and incident parallel to an optical axis on the mirror, in the direction of a sample, a deflection element for deflecting this light onto this mirror, an optic for producing a first focal point, a unit, including the mirror and the optic, in which linearly movable back and are positioned fixed in relation to one another, which is linearly movable back and forth along the optical axis, an oscillating linear drive which is mechanically connected to this unit, a sample table, movable at least in the direction of the X and Z spatial axes, for aligning the sample in relation to the first focal point, an optical arrangement for imaging a second focal point using the light originating from the sample, an aperture plate, positioned in the second focal point, for masking light originating from the sample which meets this aperture plate at a distance greater than a specific distance, a spectral filter for selecting a component of the light originating from the sample which passes through the aperture plate, and a detector for measuring the intensity of the light originating from the sample which passes through the aperture plate and is selected by the spectral filter. The optic is additionally implemented as a collimator for the light originating from the sample and the mirror is additionally implemented for deflecting this collimated light diametrically opposite to the direction of incidence of the light from the laser and parallel to the optical axis.
U.S. Pat. No. 5,260,569 also discloses a scanning light microscope in which the light emitted by a light source is aligned in parallel using a collimating lens acting as a part of the projective. The collimated light propagates in the direction parallel to the scanning direction of the microscope. Therefore, the collimated light beam—independently of the actual position of the unit movable back and forth—is always incident from the same direction on the mirror which is fixed in the unit movable back and forth. This has the consequence that the light beam is always reflected by the mirror onto the sample in the same direction and in collimated form. This collimated light is, after a 90° reflection on the mirror, bundled into a first focal point using a further projective lens which is also fixed in the unit movable back and forth. Therefore, the scanning or rasterizing of the sample may be performed using the unit movable back and forth and using the light of a light source which is attached to the unit movable back and forth. However, the attachment of the light source and photodetector outside the unit movable back and forth is preferable, so that this unit may be made simpler and lighter—to allow more rapid scanning.